Classification and Chemical

7/28/2019 Classification and Chemical

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Pure &App l . Chem. , Vol. 67, No . 4, pp . 597-600, 1995.Printed in Grsat Britain.(B 1995 IUPAC

INTERNATIONAL U NION OF PUREAND APPLIED CHEMISTRYANALYTICAL CHEMISTRY DIVISIONCOMM ISSION ON GENERAL ASPECTS OFANALYTICAL CHEMISTRY*

CLASSIFICATION AND CHEMICALCHARACTERISTICS OF IMMOBILIZEDENZYMES(Technical Report)

Prepared f o r publication byP. J . WORSFOLD

Department of Environmental Sciences, University of Plymouth, Plymouth, Devon PL4 8AA, UK

*Membership of the Commission during the preparation of this report (1991-93) was as follows:Chairman: W. E . van der Linden (Netherlands); Secretaly: C . L. Graham (UK); Titular Members:L. A. Currie (USA); St. Glab (Poland); W. Horwitz (USA); D. L. Massart (Belgium); M. Parkany(Switzerland); Associate M embers: K. Danzer (Germany); Y. Gohshi (Japan); H. Miiller (Germany);M. Otto (Germany); J. W. Stahl (US A); P. J. Worsfold (UK ); National Representatives: T. M. Tavares(Brazil); J. G araj (Czechoslovakia); J. Incz6dy (Hungary); D. Tho rbum Bum s (Ireland); R. D. Reeves(New Zealand); J. F. van Staden (RSA); F. Ingman (Sweden); S . Ate? (Turkey).Republication of this report is permitted without the need fo r form al IUPAC perm ission on condition that anacknowledgement , wi th full reference together with IUPAC copyright symbol (0 1995 IUPAC) , i s pr in ted.Publication of a translation into another language is subject to the additional condition of prior approval f rom therelevant IUPAC Narional Adhering Organiza tion.


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Classif icat ion and c hem ical character ist ics ofim m ob i l ized enzym es (Technical Repo rt)SynopsisImmobilized enzymes are becoming increasingly popular as reusable, selective analyticalchemical reagents in solid-phase flow-through reactors, a s membranes in sensors and as filmsin dry reagent kits. Classification must encom pass the properties o f the original enzyme, thetype of support used and the methods of support activation and enzyme attachment.Important characteristics of an immobilized enzyme viz a viz the enzyme in solution, e.g.,apparent activity, stability and lifetime must also be reported.1. INTRODUCTION

Immobilized enzymes have been defined as enzymes that are physically confined or localized, withretention of their catalytic activity, and which can be used repeatedly and continuously [1,2]. There is avariety of methods by which enzymes can be localized, ranging from covalent chemical bonding tophysical entrapment [1,3,4] however they can be broadly classified as follows [ 5 ] :

1. Covalent bonding o f the enzyme t o a derivatized, water-insoluble matrix.2. Intermolecular cross-linking o f enzym e molecules using multi-functional reagents.3. Adsorption of the enzyme on to a water-insoluble matrix.4. Entrapm ent of the enzyme inside a water-insoluble polym er lattice or semi-permeable membrane.

The attractions of immobilized enzymes from an analytical standpoint are primarily their reuseability,and hence cost saving, and the greater efficiency and con trol o f their catalytic activity (e.g., potentiallylonger half-lives, predictable decay rates and more efficient multi-step reactions). The immobilizedform of an enzyme can be presented f or use in three distinct forms:1. Solid-phase immobilized enzyme reactors (packed bed and open tubular) for use in continuous

flow techniques such a s flow injection analysis and post-column derivatization in liquidchromatography.2. Immobilized enzyme membranes incorporated into sensors such as potentiometric enzymeelectrodes and optical sensors.3. Solid-phase immobilized enzyme films for use in disposable, dry reagent kits with photometricdetection.The increasing use of all three forms of immobilized enzyme in analytical chemistry and the need tocompare their performance in different situations necessitates the full reporting of experimentalconditions and standard procedures for the determination and control of their properties and to aidinter-laboratory consistency of results. Immobilized microbial cells, antibodies and other biologicalmaterial (e.g . tissue slices) have also been used in each of the above application areas.

2. CLASSIFICATIONWhatever the physical form of an immobilized enzyme, i.e., solid-phase reactor, membrane or solid-phase film, there a re three important aspects of the imm obilization procedure that must be specified indetail, namely:

1. The properties of the free enzyme.2. The type of support used.3. The methods of support activation and enzyme attachment.2.1 Properties of the Free EnzymeEach enzyme has a unique systematic name, which describes the action of that enzyme, and anassociated four-number code. The first number indicates the type o f reaction that is catalyzed, the nexttwo numbers indicate the subclass and sub-subclass of reaction and the fourth number identifies theenzyme. In addition eac h enzyme has a working (trivial) name which is more commonly used, e.g., theenzyme commonly referred to as lipase has the systematic name glycerol ester hydrolase and theassociated co de number 3.2.1 .1. This classification scheme for enzymes was produced by theInternational Commission on Enzymes in 1961 [6] and is regularly updated. (The commission wasestablished in 1956 by the International Union of Biochemistw in consultation with IUPAC.) When0 1995 IUPAC, Pure and Appl ied Chemistry , 67, 4 59 8


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C/assif ication/ch emical charact erist ics o f immo bi l ized enzymes 599specifling the properties of the original enzyme its working name as well as its systematic name andassociated code number must be stated. In addition, the source of the enzyme, the physical form of th eenzyme (e.g., lyophilized), its purity (and method of purification), its catalytic activity and details ofother constituents must also be given. The above information permits direct comparison of enzymesfrom different sources.Catalytic activity is the m ost important enzyme parameter from an analytical standpoint because it has adirect bearing on sensitivity. The most widely used unit of activity is the International Unit (I.U.); oneI.U. is the amount of enzyme activity that catalyzes the transformation of one micromole of substrateper minute at 25 "C under optimal experimental conditions. How ever, the katal is the recommendedunit for enzyme activity and is defined as the amount of enzyme activity that catalyzes thetransformation of one mole of substrate per second at 25 "C under optimal experimental conditions [7].The specific activity is the number of units per milligram of protein (which is not necessarily pureenzy.ms). The molar activity (turnover number) is the number of substrate molecules transformed perminute by one mole o f enzyme when th e enzyme is the rate limiting factor.2.2 Enzyme SupportThe support material can have a critical effect on the stability of the enzyme and the efficiency ofenzyme immobilization, although it is difficult to predict in advance w hich sup port will be most suitablefor a particular enzyme. The most important requirements for a support material are that it must beinsoluble in water, have a high capacity t o bind enzyme, be chemically inert and be mechanically stable.The enzyme binding capacity is determined by the available surface area, both internal (pore size) andexternal (bead size or tube diameter), the ease with which the support can be activated and theresultant density of enzyme binding sites. The inertness refers to th e degree of non-specific adsorptionand the pH, pressure and temperature stability. In addition, the su rface charge and hydrophilicity mustbe considered. The activity of the immobilized enzyme will also depend upon the bulk mass transferand local diffusion properties of the system. In each report in this field all of these aspects must beaddressed or adequate references should be provided.Due to the often conflicting requirements of a good support, various materials have been used. Atpresent there is not a universally recommended support, the final choice being a compromise for eachparticular enzyme and experimental system. The type of support can however be convenientlyclassified into one o f three catego ries [S]:

1. Hydrophilic biopolymers based on natural polysaccharides such as agarose, dextran and cellulose.2. Lipophilic synthetic organic polymers such as polyacrylamide, polystyrene and nylon,3, Inorganic materials such as controlled pore glass and iron oxide.

Clearly the chemical structure of the support must be defined and any quantitative physical andchemical data relevant to the above factors must also be provided.2.3 Support Activation and Enzyme AttachmentAs noted earlier there a re four general approaches to enzyme immobilization. Historically entrapmentprocedures were widely used for analytical applications but currently covalent binding to a suitablyactivated support is now the most u seh l and by far the most common approach. This is therefore theonly form of imm obilization considered herein fo r classification. An activated support is defined hereinas a material having an enzyme reactive fhctional group covalently attached to an otherwise inertsurface. The stability of the resulting bond between the enzyme and the support, the local environmentof the enzyme and the potential loss o f activity on immobilization must all be considered.There is a great variety of activation procedures, detailed descriptions of which arebeyond the scope ofthis paper [1,3,5,8]. In order to classify the immobilization procedure however, details of the freeenzyme (as described above), the chemical nature of the support and the coupling reagent and theexperimental conditions used for support activation and enzyme attachment must all be stated.For polysaccharides, activation is most commonly via derivatization of available hydroxy functionsusing reagents such as cyanogen bromide [ 9 ] , triazine derivatives, e.g. cyanuric chloride, sodiumperiodate, epoxides o r benzoquinone. Polyacrylamide and its derivatives can be activated by reactionwith diamines and inorganic supports are most commonly silanized and activated by reaction with

0 1995 IUPAC, Pure and Appl ied Chemistry ,67,4


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600 COMMISSION ON GENERAL ASPECTS OF ANALYTICAL CHEMISTRY

glutaraldehyde[101. Other general schemes for support activation are diazotization or reaction withcarbodiimides, thiophosgene, thionyl chloride, N-hydroxysuccinimides or transition metal salts such astitanium chloride. The activated support is then covalently linked to the enzyme, most commonly viadirect reaction with available amino functions, e.g. on lysine residues, but also via thiol and phenolfunctions.The experimental preparation factors discussed above will have a significant effect on thecharacteristics of the immobilized enzyme, in particular its a&iyity and stability. It is thereforeimperative that the percentage of enzyme immobilized and the enzyme activity remaining afterimmobilization are stated together with the experimental conditions used for their determination.Enzyme activities for immobilized enzymes are defined in th e same way as for f ree enzymes i.e. thekatal is the recommended unit. This is the m ost important information for com paring immobilizationmethods and is often not provided. The percentage of enzyme immobilized is usually calculated bymeasuring the amount of enzyme remaining in the supernatant after immobilization and subtracting thisfrom the amount originally present. The absolute enzyme activity remaining on the support afterimmobilization is more difficu lt o determine and an apparent activity is usually measured which takesinto account mass transfer and diffusional restrictions in the experimental procedure.The o ther critical performance indicator is the stability of the immobilized enzyme with respect t o time,temperature and other storage conditions and experimental variables. Clearly this can be expressed in anumber of ways but the recommended procedure is to store the enzyme under normal operatingconditions (e.g. ambient temperature (20 "C) in an appropriate buffer) and monitor its activity afterfixed periods of time using th e same procedure as that used for determining the activity remaining afterimmobilization. For analytical purposes the effect of the controlled introduction of synthetic standards,reference materials and samples at predefined intervals and frequencies must be determined in o rder tospecifjr the minimum number of analyses possible and the lifetime of the immobilized enzyme. Theeffect of storage conditions, e.g. pH, temperature and ionic strength and of impurities incorporatedduring the immobilization step must also be considered .Other quantifiable parameters for an immobilized enzyme are its pH optimum (and working range),oxidation -reduction potential working range and the apparent Michaelis constants (KM) forappropriate substra tes (which also give an indication of immobilized enzyme selectivity). Theseparameters can be affected by immobilization and experimental conditions and therefore comprehensiveexperimental details must be provided.

4. CONCLUSIONS

3. IMMO BILIZED ENZYME CHARACTER ISTICS

Purified enzymes immobilized on solid supports by covalent binding must be classified according to theproperties of the free enzyme, the type of support used and the method of support activation andenzyme attachment. The properties of the immobilized enzyme that must be stated are the percentageof enzyme immobilized, the enzyme activity remaining after immobilization, the time and tem peraturestability of the immobilized enzyme, the pH optimum and apparent KM for appropriate substrates. Inall of the above full experimental details must be given.5. REFERENCESO.R . Zaborsky, "Immobilized Enzymes", CRC P ress, Cleveland, 1973.I. Chibata, "ImmobilizedEnzymes. Research and Development", Wiley, New York, 1978.R.A . Messing, "Immobilized Enzymes for Industrial Reactors", Academic Press, New York,1975.M.D . Trevan, "Immobilized Enzymes. An Introduction and Applications in Biotechnology",Wiley, New York, 1980.P.W . Carr and L .D . Bowers, "Immobilized Enzymes in Analytical and Clinical Chemistry.Fundamentals and Applications", Wiley, New York, 1980.International Union of Biochemistry, "Enzyme Nom enclature", Academic Press, Orlando 1984.Commission on Enzyme Nom enclature, "Enzyme Nom enclature", Elsevier, New York 1973.P. Mohr and K. Pommerening, "AffinityChromatography. Practical and Theoretical Aspects",Marcel Dekker, New York, 1985.R. Axen, J. Porath and S . Ernback, Nature, 214, 1302 (1967).H.H. W eetall, Science, 16 6, 61 5 (1969).

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